One-way PCR-based mapping of a replication initiation point (RIP).
The lamin B2 locus is the only mammalian origin whose replication initiation points (RIPs) have been mapped. Although this paper was published 8 years ago, no further mammalian RIP-mapping studies have been reported, largely due to technical difficulties of ligation-mediated (LM)-PCR used by the authors. Here, we report the development of a simple, one-way PCR-based protocol that allows one to accurately determine RIPs at mammalian origins. The procedure can be completed within 48 h from the time of cell lysis in the agarose gel. Nascent DNA is then isolated from the same gel after DNA is separated by alkaline gel electrophoresis. Subsequently, RIPs are determined by one-way PCR-based primer extension using labeled primers. Using this protocol, we have successfully mapped RIPs in the human DBF4 locus. As one-way PCR is routinely used by many scientists, this protocol will provide a powerful new tool for studying DNA replication in many organisms including mammalian cells.
Romero
Julia
J
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
Lee
Hoyun
H
D018797:Cell Cycle Proteins / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D017931:DNA Primers; D004261:DNA Replication; D004076:Digoxigenin / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D018741:Replication Origin
D018797:Cell Cycle Proteins; C485678:DBF4 protein, human; D017931:DNA Primers; D004076:Digoxigenin